Plasmacytoid dendritic cells (pDCs) produce robust type I interferons (IFN-I) within hours of viral sensing, while epithelial cells at mucosal surfaces mount a delayed response dominated by type III interferons (IFN-III). Both cell types express pattern recognition receptors that activate similar downstream transcription factors, yet they produce distinct subsets of IFNs. The mechanisms underlying these differences have remained unclear. Here, using Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) in primary human pDCs and intestinal epithelial cells, we show that IFN-I and IFN-III gene loci carry opposing, constitutively established chromatin accessibility landscapes that determine cell-type-specific interferon induction. The IFN-I locus is broadly open in pDCs and constitutively closed in epithelial cells, while the IFN-III locus displays the reciprocal pattern. Motif enrichment analysis of accessible regions at the IFN-I locus in pDCs revealed unexpected and significant enrichment of ETS family binding motifs alongside IRF motifs, which determines the cell-type-specific locus accessibility. The ETS factor PU.1 and IRF8 bind IFN-I promoters, at composite ETS-IRF elements positioned at the Positive Regulatory Domain IV (PRDIV) site of IFNB1 promoter and adjacent to the TATA-proximal IRF motif of IFNA promoters. IFNL gene promoters lack ETS recognition sequences. PU.1-IRF8 composite factor binding extends across intergenic regions of the IFN-I locus, where candidate enhancer elements marked by H3K4me1, H3K27ac, and RNA Pol II occupancy were identified. We propose that this network of ETS-IRF composite-element-anchored enhancers maintains the IFN-I locus in a constitutively poised state in pDCs, licensing the rapid and robust IFN-I response that defines pDCs. In epithelial cells, the absence of PU.1 and IRF8 renders the IFN-I locus epigenetically silent, while the IFN-III locus is constitutively open. Despite this, the delayed IFN-III gene expression in epithelial cells is due to intrinsically weaker promoter activity relative to IFN-I. These findings reveal that the divergent IFN induction of pDCs and epithelial cells is determined by the chromatin architecture prior to infection. Overall, these observations show that lineage-specific ETS-IRF regulatory factors and promoter strength determine the cell-type-specific IFN activation.

ABSTRACT In TLR7-driven macrophage activation syndrome (MAS), inflammatory hemophagocytes (iHPCs) differentiate from Ly6C HI monocytes, phagocytose red blood cells and promote disease, including anemia and thrombocytopenia. We demonstrate here that IRF5 is required for iHPC differentiation and MAS in TLR7-overexpressing (TLR7.1) mice. Both constitutive and myeloid-specific Irf5 deletion reduced iHPCs and improved anemia, thrombocytopenia and survival. Furthermore, therapeutic inhibition of IRF5 ameliorated MAS features and reduced splenic and circulating iHPCs. While cell-intrinsic IRF5 expression was required for iHPC differentiation, it was not required for TLR7.1 Ly6C HI monocyte differentiation and monocyte transcriptional programs. We further show that the transcriptome and chromatin landscape changed dramatically as iHPCs differentiated from TLR7.1 Ly6C HI monocytes. Many transcriptional programs gained in iHPCs were enriched in genes associated with IRF5-binding accessible chromatin regions, including those associated with NF-κB signaling, cytokine and chemokine production, and complement activation. Our data suggest that IRF5 collaborates with other transcription factor families, including NF-κB, ETS and AP1 members, to regulate iHPC gene programs. Together, our findings demonstrate that expression of IRF5 in myeloid cells is critical for MAS, for iHPC differentiation, and acts broadly across iHPC-specific gene programs in TLR7-driven inflammation. SUMMARY In TLR7-driven MAS, specialized monocyte-derived inflammatory hemophagocytes promote disease, including anemia and thrombocytopenia. Thulin, Lu et al. show that myeloid cell IRF5 is required for iHPC differentiation from Ly6C HI monocytes and for MAS, and that IRF5 likely partners with other transcription factors, including NF-κB, to shape iHPCs programs.

Venezuelan equine encephalitis virus (VEEV) causes encephalitis in humans and equids, and there are no vaccines or therapeutics for humans. In recent years, non-coding RNAs have emerged as critical regulatory factors affecting different cellular pathways. Specifically, long non-coding RNAs (lncRNAs) have been identified as regulators of antiviral pathways; however, their role in VEEV infection has not been assessed. Here, we show differential expression of several lncRNAs in primary mouse target cells infected with a vaccine strain of VEEV (TC-83) but not a pathogenic strain (TrD). Among the differentially expressed genes (DEGs), suppressing lncRNA small nucleolar RNA host gene 15 (Snhg15) resulted in a 7-fold increase in TC-83 replication in primary mouse astrocytes. Knockdown of Snhg15 during TC-83 infection resulted in the suppression of ten genes, all of which were also increased during TC-83 infection along with Snhg15. Most of these genes are involved in antiviral responses. KEGG pathway analysis confirmed the suppression of both pattern recognition receptor and inflammatory pathways after Snhg15 knockdown. However, Snhg15 suppression did not significantly alter NF-kB signaling in TC-83-infected cells. These data are the first to identify lncRNA responses in encephalitic alphavirus infection and demonstrate important roles for these overlooked RNAs in VEEV infection.IMPORTANCEAlthough many studies have reported differential expression of lncRNAs during viral infections, the lncRNA response to VEEV infection and its functional roles have not been previously characterized. In this study, we provide the first comprehensive analysis of host lncRNA expression in primary cells that are targeted during VEEV infection. We demonstrate that the expression of specific host lncRNAs is altered during VEEV infection and that modulation of these lncRNAs changes the expression of host antiviral and inflammatory pathways and impacts viral replication. These findings advance our understanding of VEEV-host interaction and shed light on previously unappreciated regulatory layers of infection. Given the absence of approved vaccines or antiviral therapies for VEEV, our work identifies novel host factors that may serve as potential targets for the development of anti-VEEV therapeutics upon further investigation.

Cardiopulmonary bypass (CPB) during cardiac surgery triggers inflammation that increases morbidity and mortality, though its molecular mechanisms remain unknown. To address this gap, we conducted single-nucleus RNA/ATAC sequencing (snRNA-seq/snATAC-seq) to profile transcriptional- and chromatin-level changes in circulating leukocytes from neonatal patients who underwent CPB. Classical monocytes increase after CPB, show dysregulated inflammatory genes, and exhibit altered chromatin accessibility, underscoring their role in CPB-associated inflammation. Expression of the proinflammatory cytokine interleukin-8 (IL-8/CXCL8) is significantly upregulated after CPB exposure, accompanied by increased accessibility of its promoter to AP-1 transcription factors. A genome-wide CRISPR screen in THP-1 cells identified SPTAN1 and RAF1 as novel effectors of hemodynamic stress. We further found that SPTAN1 and RAF1 activate store-operated calcium entry under CPB conditions, leading to elevated IL8 expression. We identify a shear stress-responsive SPTAN1/RAF1/store-operated calcium entry (SOCE) pathway and show that targeting it may alleviate CPB-induced inflammation, providing new insights into sterile inflammation and shear sensing in non-adherent cells.

The healthy intestine maintains homeostasis in part via immune responses to microbiota, which includes basal production of interferon cytokines. Previous work showed that Type III Interferon (IFN-λ) stimulates localized pockets of interferon-stimulated genes (ISGs) in the adult mouse intestinal epithelium at homeostasis that provide preemptive protection from viral pathogens. Here, we demonstrate that a major source of homeostatic IFN-λ production in the intestine is a population of epithelium-associated plasmacytoid dendritic cells (pDC). Depletion of bacterial microbiota in the intestine also reduces pDC abundance, and pDC depletion or bone marrow reconstitution with IFN-λ-deficient pDC results in reduced expression of homeostatic ISGs in the intestinal epithelium. Notably, intestinal pDC preferentially produce IFN-λ over Type I IFNs whereas splenic pDC produce more Type I IFNs. Comparison of intestinal and splenic pDC reveal tissue-specific changes in gene expression and genomic accessibility, including evidence of responses to transforming growth factor beta (TGF-β) in the intestine. Isolated gut pDC produce more IFN-λ than splenic pDC upon stimulation, and pre-treatment of a human pDC cell line with TGF-β results in enhanced production of IFN-λ upon stimulation. This study demonstrates that pDC are an important source of homeostatic IFN-λ in the intestine and defines the role of barrier cytokine TGF-β in regulating IFN types produced by pDC upon stimulation. Reprogramming of recruited pDC by tissue cytokines may have important implications for balancing effective antimicrobial responses with damaging inflammation at barrier tissues.

Alphaviruses (family Togaviridae) are a diverse group of positive-sense RNA (+ssRNA) viruses that are transmitted by arthropods and are the causative agent of several significant human and veterinary diseases. Interferon (IFN)-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding IFN-stimulated genes (ISGs) that are highly upregulated following viral infection and have been identified as potential restrictors of alphaviruses. The mechanism by which IFIT1 restricts RNA viruses is dependent on self and non-self-discrimination of RNA, and alphaviruses evade this recognition via their 5' untranslated region (UTR). However, the role of IFIT2 during alphavirus replication and the mechanism of viral replication inhibition is unclear. In this study, we identify IFIT2 as a restriction factor for Venezuelan equine encephalitis virus (VEEV) and show that IFIT2 binds the 3' 3'UTR of the virus. We investigated the potential role of variability in the 3'UTR of the virus affecting IFIT2 antiviral activity by studying infection with VEEV. Comparison of recombinant VEEV clones containing 3'UTR sequences derived from epizootic and enzootic isolates exhibited differential sensitivity to IFIT2 restriction in vitro infection studies, suggesting that the alphavirus 3'UTR sequence may function in part to evade IFIT2 restriction. In vitro binding assays demonstrate that IFIT2 binds to the VEEV 3'UTR; however, in contrast to previous studies, VEEV restriction did not appear to be dependent on the ability of IFIT2 to inhibit translation of viral RNA, suggesting a novel mechanism of IFIT2 restriction. Our study demonstrates that IFIT2 is a restriction factor for alphaviruses and variability in the 3'UTR of VEEV can modulate viral restriction by IFIT2. Ongoing studies are exploring the biological consequences of IFIT2-VEEV RNA interaction in viral pathogenesis and defining sequence and structural features of RNAs that regulate IFIT2 recognition.

Retinoic acid-inducible gene I (RIG-I) is a critical sensor of viral RNA and is activated in response to binding to RNA containing exposed 5'-triphosphate (5'ppp) and poly-uridine to trigger innate immune activation and response including induction of type I and III interferons (IFNs). RIG-I signaling plays a key role in not only restricting RNA virus infection but also suppressing tumor progression via oncolytic signaling. We evaluated the actions of a specific RIG-I agonist RNA (RAR) as a potential therapeutic against model tumor cell lines representing hepatocellular carcinoma (HCC). RAR constitutes a synthetic-modified RNA motif derived from the hepatitis C virus genome that is specifically recognized by RIG-I and induces innate immune activation when delivered to cells. We found that RAR directs RIG-I-dependent signaling to drive HCC cell death. Analysis of knockout cell lines lacking RIG-I, mitochondrial activator of virus signaling, or IRF3 confirmed that RAR-induced cell death signaling propagates through the RIG-I-like receptor (RLR) pathway to mediate caspase activation and HCC cell death. RAR-induced cell death is potentiated by type I IFN. Thus, RAR actions trigger HCC cell death through RIG-I linkage of RLR, caspase, and IFN signaling programs. RAR offers a potent application in antitumor therapeutic strategies leveraging innate immunity against liver cancer.

Type I interferons (IFNs) are critical for the control of viral infections, but aberrant IFN expression can result in tissue damage. RIG-I-like receptors (RLRs), such as RIG-I and MDA5, sense viral RNA and signal through the adaptor protein MAVS to induce phosphorylation and nuclear translocation of the transcription factor IRF3, thereby driving IFN production. However, activation of RLRs by endogenous RNA ligands can also induce IFNs, leading to autoinflammation. Identifying factors that suppress endogenous RNA ligands is critical for preventing IFN-induced autoimmunity. In this study, we identify a novel regulator, CELF2, that suppresses endogenous RNAs that otherwise activate the RLR pathway. We uncovered a novel role for the splicing factor CELF2 as a suppressor of immunostimulatory endogenous RNA ligands. Depletion of CELF2 in macrophages led to a spontaneous IFN and IFN-stimulated gene signature, dependent on the RIG-I-MAVS pathway. Furthermore, the transfer of RNA from CELF2-depleted macrophages was sufficient to induce type I IFN expression in naïve cells. This RNA was found to be double-stranded as RNase III treatment of RNA derived from CELF2-depleted cells ablated IFN induction in naïve cells. Immunoprecipitation of double-stranded RNA from CELF2-depleted macrophages revealed several immunostimulatory RNAs, which contribute to the increased interferon-stimulated gene signature observed in CELF2-depleted macrophages. These data indicate that CELF2 suppresses endogenous RNA ligands, which could otherwise activate RIG-I and induce an IFN signature. Overall, these findings reveal that CELF2 is an important regulator of self-RNA ligands to prevent IFN-induced autoinflammation. One-sentence summary CELF2 suppresses RIG-I-like receptor ligands that activate interferon. ### Competing Interest Statement The authors have declared no competing interest.

A cytomegalovirus (CMV) vector-based vaccine platform has emerged as a promising HIV vaccine candidate, with profound protection in preclinical animal models. Vaccine protection is highly correlated with the persistent induction of a whole-blood transcriptional signature (wbPPTS) involving immune cell, toll-like receptor (TLR), and inflammasome/death receptor signaling pathways. IL-15 was identified as the primary upstream signaling intermediate, while monocytes were identified as a major contributor of the wbPPTS. Phosphoproteomic analysis was conducted on IL-15 stimulated human monocytes over a time series (6, 12, 24, & 48hrs) to evaluate signal transduction pathway recruitment. Significant differences in phosphite abundance for key wbPPTS signaling intermediates were present in IL-15 stimulated monocytes relative to untreated controls, including MAP kinases, STAT, and IRF transcription factors. Functional enrichment analysis revealed enriched pathways linked to canonical IL-15 signaling, including AKT, mTOR, and MAPK/ERK signaling. Additionally, we observed enrichment in TLR and immune cell signaling pathways, consistent with the predicted transcriptomic signaling pathways associated with vaccine protection. These results demonstrate the diverse physiologic effects IL-15 signaling has on monocytes, with recruitment of novel signaling pathways, and have important implications for understanding the underlying protective mechanisms of the CMV vector vaccine platform. Studies supported by NIH/NIAID P01AI177688. IBA is supported by internal funds from the UW Department of Comparative Medicine. Cytokines and Chemokines and Their Receptors (CCR)

Viral-human protein interactions are critical for viral replication and modulation of the host immune response. Structural modeling of these interactions is vital for developing effective antiviral therapies and vaccines. However, 99% of experimentally determined binary host-viral interactions currently lack structural information. We aimed to address this gap by leveraging computational protein structure prediction methods. Using extensive benchmarking, we found AlphaFold to be the most accurate structure prediction model for host-pathogen protein interactions. We then predicted the structures of 11,666 binary protein interactions across 33 viral families and created the most comprehensive atomic-scale 3D viral-host protein interactomes till date ( https://3d-viralhuman.yulab.org ). By integrating these interactomes with genetic variation data, we identified population-specific signatures of selection on variants coding for interfaces of viral-human interactions. We also found that viral interaction interfaces were less conserved than non-interface regions, a striking trend that is opposite to what is observed for host interfaces, suggesting different evolutionary pressures. Systematic analyses of interface sharing between host and viral proteins binding to the same host protein revealed mutation rate-dependent differences in interface mimicry. Similar mutation rate-dependent differences were seen in the interface sharing between viral proteins binding to a host protein. We also found that the patterns of E6 protein binding to KPNA2 differed between high- and low-risk oncogenic human papillomaviruses (HPVs), and clustering based on these binding patterns allowed the classification of HPVs with unknown oncogenic risk. Our interface mimicry analyses also unveiled a novel mechanism by which herpes simplex virus-1 UL37 suppresses the antiviral immune response through disruption of the TRAF6-MAVS signalosome interaction. Overall, our comprehensive 3D viral interactomes provide a resource at unprecedented scale and resolution that will enable researchers to explore how variation and signatures of selection influence viral interactions and disease progression. This tool also facilitates the identification of conserved and unique interaction patterns across viruses, empowering researchers to generate testable hypotheses and ultimately accelerate the discovery of novel therapeutic targets and intervention strategies.

AbstractInterferon-gamma (IFNγ) is a pleiotropic cytokine produced by natural killer (NK) cells during the early infection response. IFNγ expression is tightly regulated to mount sterilizing immunity while preventing tissue pathology. Several post-transcriptional effectors dampen IFNγ expression through IFNG mRNA degradation. In this study, we identify mRNA splicing as a positive regulator of IFNγ production. While treatment with the combination of IL-12 and IL-2 causes synergistic induction of IFNG mRNA and protein, defying transcription-translation kinetics, we observe that NK cells treated with IL-12 alone transcribe IFNG with introns intact. When NK cells are treated with both IL-2 and IL-12, IFNG transcript is spliced to form mature mRNA with a concomitant increase in IFNγ protein. We find that IL-2-mediated intron splicing occurs independently of nascent transcription but relies upon NF-κB signaling. We propose that while IL-12 transcriptionally induces IFNG mRNA, IL-2 signaling stabilizes IFNG mRNA by splicing detained introns, allowing for rapid IFNγ protein production. This study uncovers a novel role for cytokine-induced splicing in regulating IFNγ through a mechanism potentially applicable to other inflammatory mediators.

Autoantibody-mediated glomerulonephritis (AGN) arises from dysregulated renal inflammation, with urgent need for improved treatments. IL-17 is implicated in AGN and drives pathology in a kidney-intrinsic manner via renal tubular epithelial cells (RTECs). Nonetheless, downstream signaling mechanisms provoking kidney pathology are poorly understood. A noncanonical RNA binding protein (RBP), Arid5a, was upregulated in human and mouse AGN. Arid5a-/- mice were refractory to AGN, with attenuated myeloid infiltration and impaired expression of IL-17-dependent cytokines and transcription factors (C/EBPβ, C/EBPδ). Transcriptome-wide RIP-Seq revealed that Arid5a inducibly interacts with conventional IL-17 target mRNAs, including CEBPB and CEBPD. Unexpectedly, many Arid5a RNA targets corresponded to translational regulation and RNA processing pathways, including rRNAs. Indeed, global protein synthesis was repressed in Arid5a-deficient cells, and C/EBPs were controlled at the level of protein rather than RNA accumulation. IL-17 prompted Arid5a nuclear export and association with 18S rRNA, a 40S ribosome constituent. Accordingly, IL-17-dependent renal autoimmunity is driven by Arid5a at the level of ribosome interactions and translation.

An important property of the host innate immune response during microbial infection is its ability to control the expression of antimicrobial effector proteins, but how this occurs post-transcriptionally is not well defined. Here, we describe a critical antibacterial role for the classic antiviral gene 2'-5'-oligoadenylate '-5 '-oligoadenylate synthetase 1 (OAS1). Human OAS1 and its mouse ortholog, Oas1b, are induced by interferon-g and protect against cytosolic bacterial pathogens such as Francisella novicida and Listeria monocytogenes in vitro and in vivo. . Proteomic and transcriptomic analysis showed reduced IRF1 protein expression in OAS1-deficient cells. Mechanistically, OAS1 binds and localizes IRF1 mRNA to the rough endoplasmic reticulum (ER)-Golgi endomembranes, licensing effective translation of IRF1 mRNA without affecting its transcription or decay. OAS1-dependent translation of IRF1 leads to the enhanced expression of antibacterial effectors, such as GBPs, which restrict intracellular bacteria. These findings uncover a noncanonical function of OAS1 in antibacterial innate immunity.

Abstract The translational arrest of protein synthesis is often a cellular response to the virus infection. However, some antiviral proteins continue to be translated through unknown mechanisms during this translational shutdown. Here, we report a mechanism by which antiviral effectors are upregulated during this translational shutdown. We found that interferon (IFN) stimulated gene, Oligoadenylate Synthetase 1 (OAS1), binds AU-rich elements (ARE) of specific mRNA, including IFNβ, prolonging the half-life and continued expression. This increased IFN expression protects from WNV infection in vitro and in vivo via downstream IFNAR signaling. This mechanism is common between human OAS1 and mouse Oas1b, independent of OAS enzyme activity and RNase L. However, human OAS1 inhibits SARS-CoV-2 replication through its canonical enzyme activity via RNase L, thus establishing two different mechanisms of OAS1 antiviral activity. These results establish OAS1 as an ARE-binding protein with a broader non-canonical function that protects IFN expression from translational shutdown.

Antiviral signaling downstream of RIG-I-like receptors (RLRs) proceeds through a multi-protein complex organized around the adaptor protein mitochondrial antiviral signaling protein (MAVS). Protein complex function can be modulated by RNA molecules that provide allosteric regulation or act as molecular guides or scaffolds. We hypothesized that RNA plays a role in organizing MAVS signaling platforms. We found that MAVS, through its central intrinsically disordered domain, directly interacted with the 3 ' untranslated regions of cellular messenger RNAs. Elimination of RNA by ribonuclease treatment disrupted the MAVS signalosome, including RNA-modulated MAVS interactors that regulate RLR signaling and viral restriction, and inhibited phosphorylation of transcription factors that induce interferons. This work uncovered a function for cellular RNA in promoting signaling through MAVS and highlights generalizable principles of RNA regulatory control of immune signaling complexes.

In response to viral infection, how cells balance translational shutdown to limit viral replication and the induction of antiviral components like interferons (IFNs) is not well understood. Moreover, how distinct isoforms of IFN-induced oligoadenylate synthetase 1 (OAS1) contribute to this antiviral response also requires further elucidation. Here, we show that human, but not mouse, OAS1 inhibits SARS-CoV-2 replication through its canonical enzyme activity via RNase L. In contrast, both mouse and human OAS1 protect against West Nile virus infection by a mechanism distinct from canonical RNase L activation. OAS1 binds AU-rich elements (AREs) of specific mRNAs, including IFNβ. This binding leads to the sequestration of IFNβ mRNA to the endomembrane regions, resulting in prolonged half-life and continued translation. Thus, OAS1 is an ARE-binding protein with two mechanisms of antiviral activity: driving inhibition of translation but also a broader, non-canonical function of protecting IFN expression from translational shutdown.

Supplementary Methods, Figure Legends 1-3 from Interleukin-15 Enhances Proteasomal Degradation of Bid in Normal Lymphocytes: Implications for Large Granular Lymphocyte Leukemias

Interleukin (IL)-4 and IL-13 are related cytokines with well-known specific roles in type 2 immune response. However, their effects on neutrophils are not completely understood. For this, we studied human primary neutrophil responses to IL-4 and IL-13. Neutrophils are dose-dependently responsive to both IL-4 and IL-13 as indicated by signal transducer and activator of transcription 6 (STAT6) phosphorylation upon stimulation, with IL-4 being more potent inducer of STAT6. IL-4-, IL-13- and Interferon (IFN)-γ-stimulated gene expression in highly purified human neutrophils induced both overlapping and unique gene expression in highly purified human neutrophils. IL-4 and IL-13 specifically regulate several immune-related genes, including IL-10, tumor necrosis factor (TNF) and leukemia inhibitory factor (LIF), while type1 immune response-related IFN-γ induced gene expression related for example, to intracellular infections. In analysis of neutrophil metabolic responses, oxygen independent glycolysis was specifically regulated by IL-4, but not by IL-13 or IFN-γ, suggesting specific role for type I IL-4 receptor in this process. Our results provide a comprehensive analysis of IL-4, IL-13 and IFN-γ -induced gene expression in neutrophils while also addressing cytokine-mediated metabolic changes in neutrophils.